In competitive binding assays, 10 mm free synthetic sialic acid (Sigma) was added 30 min before the endocytic agent and left during the assay. These were further purified by size-exclusion chromatography with elution in 50 mm KPi, 200 mm NaCl, pH 7.8, dialyzed against assay buffer (20 mm Tris, 150 mm NaCl, 2 mm tris(2-carboxyethyl)phosphine, pH 7.5), and finally concentrated to 0.5-1 mm for storage at 4 °C. For more info on sialic acid powder factory take a look at our own internet site. All 1D NMR experiments were performed using a Bruker Advance I 500-MHz spectrometer with a 5-mm PATXI 1H/D-13C/15N Z-GRD probe at 293 K.
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